The ubiquitination machinery attaches single molecules or chains of ubiquitins to substrate proteins, thus targeting them for degradation, regulating cellular localization, modulating the inflammatory response, and mediating protein-protein interactions. Ineffective removal of oncoproteins and tumor suppressors due to abnormal ubiquitination may induce malignant transformation. The relationship between the ubiquitination system and cancer is a complicated puzzle with numerous unresolved questions related to the role of ubiquitination in mediating cellular proliferation and apoptosis. The variation in lengths and linkages of the ubiquitin chains contribute to the complexity of this problem and make it a significant analytical challenge. There are seven lysine residues of ubiquitin which serve as potential poly-ubiquitin linkage sites, each with varying reactivities and each presumed to modulate different cellular pathways. The ability to map ubiquitination is the first step towards building a comprehensive understanding of the interplay between chain length, connectivity, and functional role in encoding protein fate. This proposal addresses this problem by the development of top down ultraviolet photodissociation (UVPD) mass spectrometry for identification of the distribution and linkages of ubiquitin chains on targeted proteins. The two proposed aims include: (1) Development of UVPD-MS, MS3, and middle down strategies for characterization of ubiquitin chains. Lys48- and Lys63-linked dimers and timers, and linear and Lys48-linked tetramers, pentamers and larger multimers will be analyzed by UVPD-MS and MS3 (primarily HCD followed by UVPD of selected large fragment ions) methods to evaluate the determination of linkage position and branching. NanoLC conditions will be optimized for separation and analysis of polyubiquitin mixtures. (2) Protocol testing and development on physiological targets: characterization of ubiquitinated species of p21, NF-k? inhibitor alpha, and p53. High resolution UVPD mass spectrometric analysis of three target proteins before and after proteasome inhibition (bortezomib) and deubiquitinating enzyme inhibition will be undertaken to determine the distribution of ubiquitin chain lengths and stoichiometry of the linkages. The development and application of top-down UVPD-MS will help unscramble the scope of ubiquitination of proteins and support the construction of correlations between the status of ubiquitination and implications for cancer.